1. Field
Techniques for tracking the turnover of a proteome by randomly labeling of proteins with stable isotopes 13C, 2H and 15N (carbon-13, deuterium and nitrogen-15) for the purpose of examining the dynamics of a system of proteins within an organism (e.g. a cell, whole animal or patient) in response to various drugs. The information obtained by such a method permits the determination of the organism's physiology based on the turnover and expression (i.e., relative concentration) of cellular proteins such as enzymes and membrane proteins, which often are the target of drugs or biological agents.
2. Background
The Human Genome Project has enabled the discovery of proteins, and the identities of their structures. However, the presence of a gene or its expression in a cell in the form of messenger RNA often does not correlate with the concentration of its translational product (protein), nor its function as affected by post-translational modifications. Therefore, the knowledge of a protein's concentration within a cell, its “expression” and modification, is critical to the understanding of cellular physiology and the molecular effects of therapeutic agents.
The concentration of a protein in a cell depends on the rate of its synthesis and degradation. Thus, the rate of protein turnover, the time required to synthesize a certain protein and maintain its concentration in a cell, is a sensitive indicator of cell physiology or its phenotype. The rate of protein turnover reflects a cell's response to 1) nutrient environment, 2) cell signaling due to growth factors and hormones including cytokines, 3) transcriptional regulators to differentiate or proliferate, and 4) drugs which may act in a way similar to any of the foregoing three factors.
Protein turnover is usually expressed as the rate of protein synthesis (in moles/unit time) or its half-life, which is the time required to achieve half of the maximum concentration of the protein for a given rate of protein synthesis. The determination of protein synthesis and turnover has been of great interest to biologists who are interested in understanding cellular physiology and phenotype.
Radioactive and stable isotope tracers have been used for the determination of protein synthesis and turnover for more than two decades. The basic principle of protein turnover measurement using tracers relies on the measurement of the specific activity of the “precursor” (or the labeling agent, which can be deuterium in deuterated water, or specifically labeled amino acids such as [1-13C]-leucine, [5,5,5-2H3]-leucine) and the determination of the specific activity (SA) or enrichment (E) of the labeling agent (precursor) in the protein. The newly synthesized fraction (FNP), also referred to as FNS (fraction of new synthesis), is provided by the formula:FNP=[SA or E of the precursor in protein]/[SA or E of the precursor]
Protein synthesis rate (PSR), also referred to herein as FSR (fractional synthesis rate), is calculated by dividing the quantity of new protein by the time interval for the change using the equation:PSR=[protein concentration]×(FNP)/(unit time)
When FNP or PSR are obtained for multiple time points, FNP or PSR can be plotted against time for the estimation of half-life of the turnover of the protein using compartmental analysis. An example of a rate equation for a single compartment is FNP(t)=1−FNP(max)×e−kt where k is the fraction that is cleared (turned over) per unit time. From k, t1/2 can be calculated. (see FIG. 1).
Examples of application of such a principle for the determination of protein synthesis and turnover using tracers have been published by Wolfe R R. Radioactive and Stable Isotope Tracers in Medicine. New York: Wiley-Liss, 377-416, 1992.
In order to satisfy the conditions required for calculation of FNP as previously described, the enrichment or specific activity of the labeled precursor has to be unique. The label should not be lost or gained in the process of protein synthesis, protein isolation or protein hydrolysis. Some of the more frequently used labeled amino acids are L-[1-14C]leucine or L-[1-13C]leucine, L-[ring-2H5]phenylalanine ([2H5]Phe) and L-[2H2]tyrosine ([2H2]Tyr), and L-[ring-2H4]tyrosine ([2H4]Tyr as described in Tessari P, et al., Postprandial body protein synthesis and amino acid catabolism measured with leucine and phenylalanine-tyrosine tracers. Am J Physiol Endocrinol Metab. 2003.
A labeling method using deuterated water (D2O) or heavy water in the determination of protein synthesis was introduced by, for example, Previs F., et al. Quantifying rates of protein synthesis in humans by use of 2H2O: application to patients with end-stage renal disease. Am J Physiol Endocrinol Metab 286: E665-E672, 2004 referred to herein as Previs et al., (2004) and Busch R, et al. Measurement of protein turnover rates by heavy water labeling of nonessential amino acids. Biochim Biophys Acta. 2006 May; 1760 (5):730-44 referred to herein as Busch et al. (2006) or Hellerstein.
These methods rely on the incorporation of deuterium into non-essential amino acids (NEAA) such as alanine, glycine and glutamate in living cells.
FIG. 2 shows the incorporation of deuterium or nitrogen into non-essential amino acids (NEAA) through transamination. In this figure, heavy isotopes of hydrogen (H) are shown in bold and light isotopes are shown in standard font. Since low enrichments of the isotopes are used, the heavy atom only indicates the position within the molecule that has a probability of being labeled. During the transamination process, amino acids are deaminated forming a keto-acid. The carbonyl group in the keto acid accepts a nitrogen donor from ammonium ion (15NH4+) of N-15 isotope. In subsequent reduction, the amino acid is labeled with N-15. If the reaction takes place in medium containing deuterium, the reduction process labels the amino acid with deuterium. Deuterium can also be incorporated into gluconeogenic amino acids through reduction and oxidation reactions which are not shown.
FIG. 3 shows the incorporation of 13C from [U13C6]-glucose. Glucose is a major carbon source in the synthesis of non-essential amino acids. In this figure, heavy isotopes of carbon (C) are shown in bold and light isotopes are shown in standard font. Since low enrichments of the isotopes are used, the heavy atom only indicates the position within the molecule that has a probability of being labeled. The labeling of alanine, aspartate, glutamate and glycine are illustrated. Alanine is formed from pyruvate which is a product of glycolysis. The pattern of labeling in aspartate and glutamate reflects the action of the TCA cycle. When the enrichment of 13C in glucose is low, the probability of mass isotopomers formation in these amino acids is reduced. These amino acids will contain on average mostly singly labeled (m1) species.
When cells or animals are given deuterium water, it is possible to maintain a high level of enrichment in water (0.5-2%). Previs et al. (2004) showed that plasma alanine rapidly incorporated the deuterium from water changing its CH3— group to CD3- thus increasing its molecular weight by three daltons. The actual increase in molecular weight is less due to the low level of enrichment of deuterium water used. The enrichment in alanine can be determined by gas chromatography/mass spectrometry (GC/MS) after derivatization. When a specific protein such as albumin is isolated and hydrolyzed, the enrichment in alanine isolated from the protein can be similarly determined by GC/MS. FNP can be calculated using the previously described FNP equation. The method of Hellerstein (Busch et al., 2006) differs from the method of Previs et. al. (2004) in that he used a different experimental approach to determine amino acid enrichment using mass isotopomer distribution analysis (MIDA). It should be noted that these methods are variations of the basic precursor/product enrichment ratio method previously described. They all require 1) determination of precursor SA or E, 2) isolation of specific protein of interest, 3) hydrolysis of the protein to separate the amino acids, 4) determining the enrichment in the specific amino acid of interest, and 5) application of the equations previously described. In this approach, however, any contamination of the protein in the isolated protein will alter the isotope enrichment of the amino acid in the hydrolysate therefore the accuracy of the determined FNP in such a determination is in doubt.
With the advent of high resolution maximum mass spectrometer capable of resolving molecules with molecular weights (m/z)>1000 daltons, other isotope labeling approaches have been devised to quantify proteins, determine relative protein expression and determine protein synthesis. Generally, these methods have only been applied to studies in cell culture and in yeast. The use of such methods in the quantitation of proteins has been reviewed in Beynon R J et al. Metabolic labeling of proteins for proteomics. Mol Cell Proteomics. 2005; 4(7):857-72 referred to herein as Beynon (2005).
These methods are used in the determination of (i) relative quantities (expression), (ii) protein identification, and (iii) protein turnover (synthesis).
Carbon, hydrogen, oxygen and nitrogen are the elements of organic compounds. In primitive organisms such as bacteria and yeast, organic compounds such as amino acids can be synthesized from simple molecules such as carbon dioxide and ammonia. In higher organisms such as multicellular organisms, such synthetic capability is lost. In order to introduce stable isotopes 13C, 2H and 15N (carbon-13, deuterium and nitrogen-15, respectively), other precursors such as [U13C6]-glucose, [15N, 13C]-amino acids or deuterated amino acids must be used. The application of amino acids such as L-[5,5,5-2H3]leucine results in incremental mass shifts of +3 daltons in proteins. Examples of applications of highly enriched labeled amino acids in proteomics are provided in the review by Beynon (2005).
One of the reasons for using heavily labeled amino acids such as L-[13C6]arginine to introduce stable isotopes into proteins is to separate the labeled protein from the unlabeled one by mass spectrometry such that there is no or little overlap between the spectra of these two protein species. If a lower enrichment of a fully labeled amino acid is used, it is possible to have multiple isotopomer peaks, and the information from such a spectrum is difficult to interpret.
FIG. 4 shows shifts in a peptide containing three leucines as described in Ong et al., Mol Cell Proteomics. 2002 May; 1(5):376-86 referred to herein as Ong et al. (2002). NIH3T3 cells were incubated in medium containing non-dialysed serum resulting in partially enriched d3-leucine. The mass spectrum of FIG. 4 is shown in profile of continuous distribution of molecular weights. In the synthesis of the MW 652.04 peptide, 1, 2 or 3 d3-leucines are incorporated resulting in mass shifts of +3, +6 and +9 daltons. Because the peptide in the spectrum has three positive charges, the mass shifts appear to as m/z+1, +2 and +3. However, when cells were incubated with 99% enriched d3-leucine with dialysed serum, only +9 molecular species was observed. (see FIG. 5) The mass spectrum of this peptide is complicated by the existence of an isotope envelope due to natural abundance of 13C, 18O and 15N and by contamination by other peptides.
Stable isotope labeling with amino acids in cell culture (SILAC) is a technique for labeling proteins with a labeled essential amino acid for the determination of protein expression (relative concentration of proteins in experimentally treated and control cells). In the SILAC method, proteins are completely labeled in cell cultures using fully labeled essential amino acids (for example d3-leucine, 15N-13C-arginine, etc. (www.silac.org)). Cells are grown for several days (after several cell divisions) until the corresponding essential amino acid in proteins is completely replaced with the labeled essential amino acids. These fully labeled proteins are then used as reference standard to determine changes in protein expression in these cells after molecular manipulation. This approach allows the determination of changes in protein expression levels (concentrations) of many cellular proteins by determining the mass spectral peaks corresponding to the unlabeled (from manipulated cells) to labeled (from control) protein. Protein expression is provided by the following formula:Protein expression=[unlabeled peak]/[labeled peak]
A ratio of one means that a protein is neither under nor over expressed. A ratio of <1 means under-expression (concentration is less than that of the control) and >1, over-expression (concentration is greater than that of the control).
FIG. 5 shows mass shifts in a peptide containing d3-leucine (from Ong et al. 2002). NIH3T3 cells were incubated in medium containing 99% d3-leucine and dialysed serum for 24 hour. In the synthesis of the MW 652.04 peptide, d3-leucines are incorporated resulting in a mass shift of m/z+3 in the spectrum (actually +9 daltons as previously discussed). In subsequent discussion, this peak is designated as d3. In 24 hours, some unlabeled (unenriched) peptide remained. By adding a known amount of protein from cells grown in [5,5,52H3] leucine, the relative protein expression is given by the ratio of d3/d0 when the d0 and d3 peaks are compared as the “unlabeled” and the “labeled” peaks. The other relevant peaks of the spectrum are ignored. Since the exact precursor enrichment is not known, protein synthesis cannot be determined.
Attempts have been made to determine protein synthesis in vivo using mass isotopomer distribution analysis (MIDA) as an extension of the same method in the determination of synthesis of polymers. See, for example, Papageorgopoulos C, et al. Measuring protein synthesis by mass isotopomer distribution analysis (MIDA). Anal Biochem. 1999 Feb. 1; 267(1):1-16 and Hellerstein M K, Neese R A. Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations. Am J. Physiol. 1999 June; 276(6 Pt 1):E 1146-70. Doherty M K et al. Proteome dynamics in complex organisms: using stable isotopes to monitor individual protein turnover rates. Proteomics. 2005 February; 5(2):522-33.
In these demonstrations, rats were infused with [5,5,5-2H3]leucine (99% enriched) via the jugular catheter for 24 h using a minipump at a rate of ˜50 mg/kg/h. Muscle was harvested and creatine kinase (CK) was isolated. Trypsin digest of the protein was analysed using an electrospray ionization/magnetic sector mass spectrometer. Mass isotopomers containing leucine isotope in peptides rich in leucine was determined. Incorporation of [5,5,5-2H3]leucine would result in mass shift of +3, +6, etc. depending on the number of leucine in the peptide and the enrichment of intramyocyte leucine. However, due to the low protein turnover rate, the +3 or +6 isotopomers were not detected. Even though MIDA method is theoretically possible in such application, the feasibility of the MIDA method as described by Papageorgopoulos C, Caldwell K, Schweingrubber H, Neese R A, Shackleton C H, Hellerstein M. Measuring synthesis rates of muscle creatine kinase and myosin with stable isotopes and mass spectrometry. Anal Biochem. 2002 Oct. 1; 309(1): 1-10 referred to herein as Papageorgopoulos et al. (2002) for in vivo study was not demonstrated.
Determination of low levels of deuterium incorporation into peptides from deuterated water was recently described by Wang et al. (Wang B, Sun G, Anderson D R, Jia M, Previs S, Anderson V E. Isotopologue distributions of peptide product ions by tandem mass spectrometry: quantitation of low levels of deuterium incorporation. Anal Biochem. 367(1):40-8, 2007) by determining excess molar ratio at M1 (peptide with one deuterium incorporated). However, such an approach using excess mass calculation is similar to the excess M1 calculation of Hellerstein (Hellerstein M K. Relationship between precursor enrichment and ratio of excess M2/excess M1 isotopomer frequencies in a secreted polymer. Biol. Chem. 266(17):10920-4, 1991), and cannot be used to determine protein synthesis without additional information of the isotopomer distribution of the new peptide.
Another method using mass spectrum for the determination of protein synthesis is that of Cargile B J, et al. (Synthesis/degradation ratio mass spectrometry for measuring relative dynamic protein turnover. Anal Chem. 2004 Jan. 1; 76(1):86-97) referred to herein as Cargile et al. (2004). The method introduces 13C carbon into protein by substituting natural glucose with [U13C6]-glucose (final enrichment>50%). In organisms which can synthesize essential and non-essential amino acids from glucose and nitrogen, [U13C6]-glucose effectively replaces 12C by 13C in protein creating a heavy protein which can be separated by mass spectrometry. By quantitating the intensity of the labeled and the unlabeled peaks, a synthesis/degradation ratio can be calculated to represent relative dynamic protein turnover. Such a method is useful for the study of organisms such as bacteria and yeast, which can synthesize their amino acids from glucose and nitrogen.
FIG. 6 shows the labeling of protein with highly enriched [U13C6]glucose (see Ong et al., 2002) E. coli strain was grown in minimal medium with [U13C6]glucose as the carbon source. MALDI-TOF/TOF spectrum of peptide VEGGQHLNVMVLR [SEQ ID NO: 1] shows well separated labeled and unlabeled peaks of 12C and 13C peptides. The mathematical model (approximation with a Poisson distribution) is applicable only with 12C and 13C peptides, i.e., the model does not resolve overlapping ions.